An understanding of the concentration dependence of third-strand binding is an important prerequisite for the planning of future experiments, whether in vitro or in vivo. Experiments in which the third-strand:plasmid molar ratio is varied provide the KD of binding as well as saturation conditions. Since third-strand binding could be influenced by an adjacent bound third-strand, titration experiments will be performed on both single independent and contiguous repetitive targets. At this point, the goal is to determine the basic equilibrium constants of third-strand binding to targets in a supercoiled plasmid. By using single-copy and contiguous multi-copy targets, the cooperativity of binding, if any, can be determined.